Major Immediate-early Research Articles

Effects of human cytomegalovirus immediate-early proteins on p53-mediated apoptosis in coronary artery smooth muscle cells.

Restenotic and atherosclerotic lesions often contain smooth muscle cells (SMCs), which display high rates of proliferation and apoptosis. Human cytomegalovirus (HCMV) may increase the incidence of restenosis and predispose to atherosclerosis. Although the mechanisms contributing to these processes are unclear, studies demonstrate that one of the immediate-early (IE) gene products of HCMV, IE2-84, binds to and inhibits p53 transcriptional activity. Given the role of p53 in mediating apoptosis, we studied the ability of IE2-84 to inhibit p53-dependent apoptosis in human coronary artery SMCs. Apoptosis of SMCs was induced either by use of an adenovirus vector encoding human wild-type p53 protein or by treatment with doxorubicin. HCMV IE1-72 and IE2-84, the major IE proteins of HCMV, were overexpressed separately with adenovirus vectors encoding each protein, and the effects on p53-induced apoptosis were examined by both nick end-labeling (TUNEL) assay and flow cytometry. Expression of IE2-84, but not IE1-72, protected SMCs from p53-mediated apoptosis. These data indicate that an HCMV IE protein antagonizes p53-mediated apoptosis, suggesting a pathway by which HCMV infection predisposes to SMC accumulation and thereby contributes to restenosis and atherosclerosis.

Effects of human cytomegalovirus major immediate-early proteins in controlling the cell cycle and inhibiting apoptosis: studies with ts13 cells.

The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) encodes several MIE proteins (MIEPs) produced as a result of alternative splicing and polyadenylation of the primary transcript. Previously we demonstrated that the HCMV MIEPs expressed from the entire MIE gene could rescue the temperature-sensitive (ts) transcriptional defect in the ts13 cell line. This defect is caused by a ts mutation in TAFII250, the 250-kDa TATA binding protein-associated factor (TAF). These and other data suggested that the MIEPs perform a TAF-like function in complex with the basal transcription factor TFIID. In addition to the transcriptional defect, the ts mutation in ts13 cells results in a defect in cell cycle progression which ultimately leads to apoptosis. Since all of these defects can be rescued by wild-type TAFII250, we asked whether the MIEPs could rescue the cell cycle defect and/or affect the progression to apoptosis. We have found that the MIEPs, expressed from the entire MIE gene, do not rescue the cell cycle block in ts13 cells grown at the nonpermissive temperature. However, despite the maintenance of the cell cycle block, the ts13 cells which express the MIEPs are resistant to apoptosis. MIEP mutants, which have previously been shown to be defective in rescuing the ts transcriptional defect, maintained the ability to inhibit apoptosis. Hence, the MIEP functions which affect transcription appear to be separable from the functions which inhibit apoptosis. We discuss these data in the light of the HCMV life cycle and the possibility that the MIEPs promote cellular transformation by a "hit-and-run" mechanism.

Murine Cytomegalovirus Immediate-Early Promoter Directs Astrocyte-Specific Expression in Transgenic Mice

Murine cytomegalovirus (MCMV), which causes acute, latent, and persistent infection of the natural host, is used as an animal model of human cytomegalovirus (HCMV) infection. Transcription of MCMV immediate-early (IE) genes is required for expression of the early and late genes and is dependent on host cell transcription factors. Cell-type-specific expression activity of the MCMV IE promoter was analyzed in transgenic mice generated with the major IE (MIE) enhancer/promoter involving nucleotides -1343 to -6 (1338 bp) connected to the reporter gene lacZ. Distinct expression was observed in the brain, kidneys, stomach, and skeletal muscles. Weak expression was observed in a portion of the parenchymal cells of the salivary glands and pancreas, and expression was hardly detected in the lungs, intestine, or immune and hematopoietic organs such as the thymus, spleen, lymph nodes, and bone marrow. The spectrum of organs positive for expression was narrower than that of the HCMV MIE promoter-lacZ transgenic mice reported previously and showed a greater degree of cell-type specificity. Interestingly, astrocyte-specific expression of the transgene was observed in the brain and primary glial cultures from the transgenic mice by combination of beta-galactosidase (beta-Gal) expression and immunostaining for cell markers. However, the transgene was not expressed in neurons, oligodendroglia, microglia, or endothelial cells. Furthermore, the beta-Gal expression in glial cultures was stimulated significantly by MCMV infection or by addition of calcium ionophore. These observations indicated that expression activity of the MCMV IE promoter is strictly cell-type specific, especially astrocyte-specific in the brain. This specific pattern of activity is similar to that of natural HCMV infection in humans.

Sequence variation within three important cytomegalovirus gene regions in isolates from four different patient populations.

We determined the nucleotide (nt) and amino acid (aa) heterogeneities of three distinct regions of the human cytomegalovirus (CMV) genome for 46 low-passage CMV isolates from four different patient populations (congenitally infected infants, children attending day-care centers, renal transplant recipients, and human immunodeficiency virus-infected individuals) and for two laboratory strains (CMV Ad169 and Towne). The gene regions for the major immediate-early (MIE) exon 4 gene (nt positions 1702 to 1982, aa positions 152 to 244), the DNA polymerase gene (nt positions 2797 to 3046, aa positions 713 to 795), and the glycoprotein B (gB) gene (nt positions 1698 to 1884, aa positions 567 to 628) were sequenced. The sequence information was used to design sets of nested PCR primers directed against the most highly conserved regions identified. MIE was the most variable gene region compared to the variability of the DNA polymerase and gB gene regions. Comparison of the sequences of all 46 isolates with that of Ad169 revealed nt and aa sequence homologies of 87.9 and 87.2%, respectively, within the MIE gene compared to 92.8 and 100% homologies, respectively, within the DNA polymerase gene and 93 and 95.2% homologies, respectively, within the gB gene. Within the MIE gene, compared to the Ad169 nt sequence the homology at the nt level among isolates obtained from children attending day-care centers was high (96.4%), while it was lower (90%) among isolates obtained from the other three patient populations. Preliminary results of a nested PCR with oligonucleotide primers selected from the DNA polymerase gene region with a low level of nt sequence variation indicates that primers selected from this region might be more powerful for use in PCR than primers selected from the MIE gene region.

A cis repression sequence adjacent to the transcription start site of the human cytomegalovirus US3 gene is required to down regulate gene expression at early and late times after infection.

Human cytomegalovirus has two enhancer-containing immediate-early (IE) promoters with a cis repression sequence (CRS) positioned immediately upstream of the transcription start site, designated the major IE (MIE) promoter and the US3 promoter. The role of the CRS upstream of the US3 transcription start site in the context of the viral genome was determined by comparing the levels of transcription from these two enhancer-containing promoters in recombinant viruses with a wild-type or mutant CRS. Upstream of the CRS of the US3 promoter was either the endogenous enhancer (R2) or silencer (R1). The downstream US3 gene was replaced with the indicator gene chloramphenicol acetyltransferase (CAT). Infected permissive human fibroblast cells or nonpermissive, undifferentiated monocytic THP-1 cells were analyzed for expression from the US3 promoter containing either the wild-type or mutant CRS. With the wild-type CRS, the maximum level of transcription in permissive cells was detected within 4 to 6 h after infection and then declined. With the mutant CRS and the R2 enhancer upstream, expression from the US3 promoter continued to increase throughout the viral replication cycle to levels 20- to 40-fold higher than for the wild type. In nonpermissive or permissive monocytic THP-1 cells, expression from the US3 promoter was also significantly higher when the CRS was mutated. Less expression was obtained when only the R1 element was present, but expression was higher when the CRS was mutated. Thus, the CRS in the enhancer-containing US3 promoter appears to allow for a short burst of US3 gene expression followed by repression at early and late times after infection. Overexpression of US3 may be detrimental to viral replication, and its level of expression must be stringently controlled. The role of the CRS and the viral IE86 protein in controlling enhancer-containing promoters is discussed.

The Maastricht Strain and England Strain of Rat Cytomegalovirus Represent Different Betaherpesvirus Species Rather Than Strains

The major immediate early (MIE) locus of the Maastricht strain of rat cytomegalovirus (RCMV) was found to comprise five exons of which the first is noncoding. The first three exons are spliced to either exon 4, generating IE1, or exon 5, generating IE2. An additional splicing event unique to RCMV (Maastricht) was identified in exon 5, resulting in a 466-bp deletion. IE1 transcripts were detected exclusively during the IE phase of infectionin vitro,whereas IE2 transcripts were detected during both the IE and late phase of infection. The similarities between amino acid sequences derived from the MIE gene of RCMV (Maastricht) and murine cytomegalovirus are low (22 and 37% for IE1 and IE2, respectively). Surprisingly, the similarities between the MIE proteins of RCMV (Maastricht) and the England strain of RCMV are also low (23 and 32% for IE1 and IE2, respectively). This suggests that these RCMV strains represent different betaherpesvirus species rather than strains. This is underscored by the difference between both viruses in genome size as well as growth characteristics. The existence of two different RCMV-like species might have important implications for the use of these viruses as models for human cytomegalovirus.

Evaluation and mapping of the DNA binding and oligomerization domains of the IE2 regulatory protein of human cytomegalovirus using yeast one and two hybrid interaction assays

The 86-kDa IE2 nuclear phosphoprotein encoded by the human cytomegalovirus (HCMV) major immediate-early (MIE) gene behaves as both a non-specific transactivator of viral and cellular gene expression and as a specific DNA-binding protein targeted to the cis-repression sequence (CRS) at the cap site of its own promoter/enhancer region. Although the IE2 protein produced in bacteria has been shown to bind to the 14-bp palindromic CRS motif and IE2 synthesized in vitro forms stable dimers in solution through the conserved C-terminus of the protein, there is no direct evidence as yet that the intracellular mammalian forms of IE2 do so. Here, we show that the intact HCMV IE2 protein both binds to CRS DNA and dimerizes in yeast cells. In a one-hybrid assay system, a GAL4/IE2 fusion protein expressed in yeast cells activated target HIS3 expression only when CRS sites were located upstream of the GAL1 minimal promoter, but failed to do so on mutant CRS sites, demonstrating a requirement for sequence-specific DNA-binding by IE2. Examination of a series of deletion and triple amino acid point mutations in the C-terminal half of IE2 mapped the domains required for DNA-binding in yeast to the entire region between codons 313 and 579, whereas in the previous in vitro study with truncated bacterial GST fusion proteins, it was mapped to between codons 346 and 579. Transient co-transfection assays with deleted IE2 effector genes in Vero cells showed that the extra segment of IE2 between codons 313 and 346 is also required for both autoregulation and transactivation activity in mammalian cells. In a two-hybrid assay to study IE2 self-interations, we generated both GAL4 DNA-binding (DB) and activation domain (A)/IE2 fusion proteins and showed that IE2 could also dimerize or oligomerize through the C-terminus of the protein in yeast cells. Domains required for this interaction were all mapped to within the region between codons 388 and 542, which is coincident with the domain mapped previously for dimerization by co-translation and immunoprecipitation in vitro. Comparison of the domains of the IE2 protein required for CRS binding and dimerization in yeast suggests that these activities correlate precisely with requirements for the negative autoregulation function of the IE2 protein in mammalian cells.

Evaluation of PCR Primers for Early Diagnosis of Cytomegalovirus Infection following Liver Transplantation

The availability of microbiologic methods that detect early replication of cytomegalovirus (CMV) posttransplantation will enhance the process of initiating preemptive antiviral therapy prior to the appearance of CMV disease. Using PCR techniques we sought to determine which region of the CMV genome present in peripheral blood leukocytes (PBLs) or serum provides the highest sensitivity for the detection of CMV posttransplantation. Blood samples were prospectively collected weekly for at least 8 weeks from a cohort of 21 consecutive liver transplant recipients not receiving anti-CMV prophylaxis. Results of PCR assays were correlated with recovery of CMV in cell cultures and histopathological findings from biopsy specimens of infected organs to assess clinical symptomatic infection. Of 148 specimens, primer pairs directed to the HindIII-X fragment region of CMV detected target DNA with a 94% sensitivity, compared to an 87% sensitivity with primer pairs directed to EcoRI fragment D, 32% sensitivity with primer pairs directed to the immediate-early antigen 1 gene (IEA1 gene), and 20% sensitivity with primer pairs directed to the major immediate-early (MIE) gene. The performance characteristics in terms of the sensitivity of primers for amplifying CMV DNA associated with symptomatic infection ranged from 100% (HindIII-X) to 20% (MIE gene); however, specificity was inversely related (HindIII-X, 45%; MIE gene, 91%) to primers directed to these gene targets. When HindIII-X and EcoRI-D primer sets were used, CMV DNA from PBLs was a more sensitive target than CMV DNA from serum for the early detection of symptomatic CMV infection (17 versus 12 days). Importantly, CMV DNA was not detected in five patients with no evidence of this viral infection. In conclusion, primers directed to the HindIII-X fragment region were the most optimal for the early detection of CMV DNA in PBLs and sera from symptomatic liver transplant recipients.

Diagnosis of congenital and perinatal infection by cytomegalovirus using polymerase chain reaction

The practical application of a polymerase chain reaction (PCR) amplification for the diagnosis of congenital and perinatal cytomegalovirus (CMV) infections was evaluated. Three hundred five urine samples were tested by PCR and conventional virus isolation in cell culture. Viruria was detected in 47 urine samples by PCR using a primer pair which amplifies part of the major immediate-early (MIE) CMV genome. The PCR compared to virus isolation showed 89.6% sensitivity, 98.5% specificity and 91.5% positive predictive value. PCR with primer pairs amplifying parts of the glycoprotein B and glycoprotein H genes of CMV were used for confirmation of the positivity of the 47 urine samples. We concluded that this CMV PCR assay in urine has a suitable sensitivity for the diagnosis of congenital and perinatal infections and its specificity is highly increased by use of more than one pair of primers among the ones we used.

Herpes simplex virus type 1 immediate early gene expression is stimulated by inhibition of protein synthesis.

Herpes simplex virus type 1 (HSV-1) transcription can be arrested at the immediate early (IE) stage by continuous treatment of cells with inhibitors of protein synthesis, usually cycloheximide, from the time of infection. We have analysed the effect of cycloheximide on IE gene expression with HSV-1 mutants deficient in the production of functional levels of the three major transactivators, the virion protein (VP16) and two IE proteins (ICP0 and ICP4). Expression from the HSV-1 IE promoters that control synthesis of ICP0 and ICP27 was, unexpectedly, stimulated by inhibition of protein synthesis. The effect was observed for the ICP0 promoter in its normal genome location and also when cloned upstream of the Escherichia coli lacZ coding sequences and inserted into the viral thymidine kinase locus. Expression from the human cytomegalovirus major IE promoter, when cloned into the genome of HSV-1 mutants, was also increased by inhibition of protein synthesis. Cycloheximide did not affect the intracellular stability of lacZ-specific RNA, suggesting that the response represented an increase in mRNA production. Activation of the ICP0 promoter was observed when protein synthesis was blocked by alternative agents. Since inhibitors of protein synthesis are known to activate cellular signal transduction pathways, our findings demonstrate new mechanisms for the regulation of HSV-1 IE gene expression which may be important during latency and reactivation. The results also highlight previously unrecognized difficulties in analysing the intrinsic activities of promoters when cloned into the HSV-1 genome.

Transcription factor Sp1 mediates cell-specific trans-activation of the human cytomegalovirus DNA polymerase gene promoter by immediate-early protein IE86 in glioblastoma U373MG cells.

Human cytomegalovirus (HCMV) gene expression is highly cell and tissue specific. Cell factor-mediated regulatory interactions are involved in regulating the restricted expression of the HCMV major immediate-early (IE) gene (J. F. Baskar, P. P. Smith, G. Nilaver, R. A. Jupp, S. Hoffmann, N. J. Peffer, D. J. Tenney, A. M. Colberg-Poley, P. Ghazal, and J. A. Nelson, 70:3207-3213, 1996). To gain an understanding of HCMV early gene activation, we studied the effect of each of the three major IE proteins, IE72, IE86, and IE55, on the HCMV DNA polymerase gene (pol; UL54) promoter. In transient-expression assays, the IE86 protein alone was able to transactivate the pol promoter, but IE72 and IE55 were not, in permissive U373MG cells. However, we were unable to detect IE86-mediated transactivation in nonpermissive HeLa or C33-A cells. Using electrophoretic mobility shift assays (EMSAs), we found that expression of the IE86 protein in U373MG cells resulted in specific binding of a DNA complex to an inverted-repeat element, IR1, of the pol promoter. Antibody supershifting and EMSA-Western blotting experiments further showed that IE86 and the cellular transcription factor Sp1 were components of the IR1 DNA-binding complex. Furthermore, we found that binding of DNA by Sp1 was dramatically increased in the presence of IE86. Interestingly, this IE86-induced DNA-binding activity of Sp1 was inhibited by a repressor activity presented in HeLa cells. In summary, our study suggests that a viral regulatory protein can modulate the DNA binding activity of a cellular transcription factor, resulting in cell-specific transactivation of viral genes.

The human polyomavirus BK T antigen induces gene expression in human cytomegalovirus

Co-infections or co-habitations of cells by two or more viruses may occur in the human organism. Human cytomegalovirus (HCMV) and the human polyomavirus BK (BKV) have common host cells and may both establish lifelong latency/persistence following primary infection. Both viruses may become reactivated by immunosuppression or other conditions which upset host–virus balance, and they encode gene products with the inherent potential of acting as heterologous transacting factors for expression of cellular or viral genes. It has been shown that HCMV induces gene expression and replication of primate polyomaviruses. We now demonstrate that BKV is able to enhance the expression of HCMV immediate early (IE1 and 2) as well as the early (E) protein pp65 during double infections in semi-permissive cells. By transfection experiments it was established that the phenomenon is due to heterologous transcriptional transactivation of the HCMV major IE promoter (MIEP) by the BKV large T antigen, without contribution from the small t antigen.

Negative Regulation of a Heterologous Promoter by Human Cytomegalovirus Immediate-Early Protein IE2

The HCMV IE2 protein promiscuously activates transcription of many viral and cellular genes. IE2 also negatively autoregulates its own expression by binding to a strategically positioned IE2 binding site, called CRS, located immediately downstream of the TATA box of the HCMV major IE promoter. Here we show that IE2 is able to repress transcription driven by a heterologous promoter, RSV LTR. Repression of RSV LTR by IE2 is completely dependent of DNA sequences downstream of the TATA box of RSV LTR. A DNA sequence, 5′-CGATACAATAAACG-3′, evidently matching the proposed CRS consensus sequence, is located between nucleotides −13 and +1 (relative to the transcription start site) of RSV LTR. Three lines of evidence support the notion that this RSV CRS element is involved in the IE2-mediated repression of RSV LTR. First, introduction of mutation to the RSV CRS element renders to the mutant RSV LTR resistance to IE2-mediated repression. Second, a mutant IE2 defective in DNA binding cannot downregulate transcription from RSV LTR. Third, IE2 specifically binds to the wild-type, but not the mutant, RSV CRS elementin vitro.These data, in conjunction with previous works, demonstrate that IE2 can passively repress transcription of homologous and heterologous promoters that contain a CRS element.

TAF-like functions of human cytomegalovirus immediate-early proteins.

The human cytomegalovirus (HCMV) major immediate-early (IE) proteins IEP86 (IE2(579aa)) and IEP72 (IE1(491aa)) can transcriptionally activate a variety of simple promoters containing a TATA element and one upstream transcription factor binding site. In our previous studies, transcriptional activation was shown to correlate with IEP86 binding to both the TATA-box binding protein (TBP) and the transcription factor bound upstream. IEP72 often synergistically affects the activation by IEP86, although it has not previously been shown to directly interact in vitro with IEP86, TBP, or transcription factors (e.g., Sp1 and Tef-1) bound by IEP86. We report biochemical and genetic evidence suggesting that the major IE proteins may perform a function similar to that of the TBP-associated factors (TAFs) which make up TFIID. Consistent with this model, we found that the major IE proteins interact with a number of TAFs. In vitro, IEP86 bound with drosophila TAF(II)110 (dTAF(II)110) and human TAF(II)130 (hTAF(II)130), while IEP72 bound dTAF(II)40, dTAF(II)110, and hTAF(II)130. Regions on major IE proteins which mediate binding have been defined. In addition, our data indicate that both IEP72 and IEP86 can bind simultaneously to hTAF(II)130, suggesting that this TAF may provide bridging interactions between the two proteins for transcriptional activation and synergy. In agreement, a transcriptional activation mutant of IEP72 is unable to participate in bridging. Confirmation that these in vitro interactions were relevant was provided by data showing that both IEP72 and IEP86 copurify with TFIID and coimmunoprecipitate with purified TFIID derived from infected cell nuclei. To further support a TAF-like function of the IE proteins, we have found that the IE proteins expressed from the intact major IE gene, and to a lesser extent IEP86 alone, can rescue the temperature-sensitive (ts) transcriptional defect in TAF(II)250 in the BHK-21 cell line ts13. Analyses of mutations in the major IE region show that IEP86 is essential for rescue and that IEP72 augments its effect, and that mutations which affect TAF interactions are debilitated in rescue. Our data, showing that the IE proteins can bind with TFIID and rescue a ts transcriptional defect in TAF(II)250, support the model that the IE proteins perform a TAF-like function as components of TFIID.

Repression of gene expression upon infection of cells with herpes simplex virus type 1 mutants impaired for immediate-early protein synthesis.

Herpes simplex virus type 1 (HSV-1) mutants defective in immediate-early (IE) gene expression do not readily enter productive replication after infection of tissue culture cells. Instead, their genomes are retained in a quiescent, nonreplicating state in which the production of viral gene products cannot be detected. To investigate the block to virus replication, we used the HSV-1 triple mutant in1820K, which, under appropriate conditions, is effectively devoid of the transactivators VP16 (a virion protein), ICP0, and ICP4 (both IE proteins). Promoters for the HSV-1 IE ICP0 gene or the human cytomegalovirus (HCMV) major IE gene, cloned upstream of the Escherichia coli lacZ coding sequences, were introduced into the in1820K genome. The regulation of these promoters and of the endogenous HSV-1 IE promoters was investigated upon conversion of the virus to a quiescent state. Within 24 h of infection, the ICP0 promoter became much less sensitive to transactivation by VP16 whereas the same element, when used to transform Vero cells, retained its responsiveness. The HCMV IE promoter, which is not activated by VP16, also became less sensitive to the HCMV functional homolog of VP16. Both elements remained available for transactivation by HSV-1 IE proteins at 24 h postinfection, showing that the in1820K genome was not irreversibly inactivated. The promoters controlling the HSV-1 ICP4, ICP22, and ICP27 genes also became essentially unresponsive to transactivation by VP16. The ICP0 promoter was induced when hexamethylene bisacetamide was added to cultures at the time of infection, but the response to this agent was also lost by 24 h after infection. Therefore, promoter elements within the HSV-1 genome are actively repressed in the absence of IE gene expression, and repression is not restricted specifically to HSV-1 IE promoters.

Human cytomegalovirus IE2 86-kilodalton protein binds p53 but does not abrogate G1 checkpoint function.

Physical interactions between human cytomegalovirus (HCMV) immediate-early (IE) proteins and key cell cycle regulatory proteins have been suggested as a mechanism whereby this herpesvirus modifies cellular control of proliferation. Observed similarities to interactions of other DNA virus proteins (human papillomavirus type 16 E6 and E7, simian virus 40 large T antigen, and adenovirus type 5 E1A and E1B) with cell cycle modulatory proteins such as p53 and Rb have suggested that HCMV IE proteins may likewise alter the G1-to-S phase transition. The IE2 region gene product IE86 has been shown to specifically bind p53, potentially modifying p53 G1 checkpoint function. To examine this possibility, p53-mediated G1 arrest in the presence of IE86 was assessed. Retroviral constructs were created to facilitate the stable expression of IE86 and IE72, another IE protein implicated in HCMV-mediated alteration of cell cycle progression. Western analysis and immunoprecipitation confirmed IE protein expression and binding of IE86 to p53, respectively. Chloramphenicol acetyltransferase assays examining the ability of IE86 to repress activity from the HCMV major IE promoter or activate the HCMV early promoter for the 2.2-kb class of RNAs demonstrated the functional integrity of the IE86 protein. Induction of DNA damage in normal, uninfected fibroblasts (FB) or FB expressing IE86 by actinomycin D (Act D) resulted in increased p53 levels, a predominance of the hypophosphorylated form of Rb, and increased expression of both p21(CIP1/WAF1) and mdm-2. Fluorescence-activated cell sorting revealed that both uninfected and IE86-expressing FB experienced dramatic G1 arrest following exposure to Act D. The clear demonstration of these p53-dependent responses in the presence of IE86 indicates that binding to this viral protein does not compromise the ability of p53 to elicit growth arrest following DNA damage.

The major immediate-early proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML-associated nuclear bodies at very early times in infected permissive cells.

The major immediate-early (MIE) gene products of human cytomegalovirus (HCMV) are nuclear phosphoproteins that are thought to play key roles in initiating lytic cycle gene regulation pathways. We have examined the intranuclear localization pattern of both the IE1 and IE2 proteins in virus-infected and DNA-transfected cells. When HCMV-infected human diploid fibroblast (HF) cells were stained with specific monoclonal antibodies, IE1 localized as a mixture of nuclear diffuse and punctate patterns at very early times (2 h) but changed to an exclusively nuclear diffuse pattern at later times. In contrast, IE2 was distributed predominantly in nuclear punctate structures continuously from 2 to at least 12 h after infection. These punctate structures resembled the preexisting PML-associated nuclear bodies (ND10 or PML oncogenic domains [PODs]) that are disrupted and dispersed by the IE110 protein as a very early event in herpes simplex virus (HSV) infection. However, HCMV differed from HSV by leading instead to a change in both the PML and SP100 protein distribution from punctate bodies to uniform diffuse patterns, a process that was complete in 50% of the cells at 2 h and in 90% of the cells by 4 h after infection. Confocal double-label indirect immunofluorescence assay analysis confirmed that both IE1 and IE2 colocalized transiently with PML in punctate bodies at very early times after infection. In transient expression assays, introduction of IE1-encoding plasmid DNA alone into Vero or HF cells produced the typical total redistribution of PML into a uniform nuclear diffuse pattern together with the IE1 protein, whereas introduction of IE2-encoding plasmid DNA alone resulted in stable colocalization of the IE2 protein with PML in the PODs. A truncated mutant form of IE1 gave large nuclear aggregates and failed to redistribute PML, and similarly a deleted mutant form of IE2 failed to colocalize with the punctate PML bodies, confirming the specificity of these effects. Furthermore, both Vero and U373 cell lines constitutively expressing IE1 also showed total PML relocalization together with the IE1 protein into a nuclear diffuse pattern, although a very small percentage of the cells which failed to express IE1 reverted to a punctate PML pattern. Finally, the PML redistribution activity of IE1 and the direct association of IE2 with PML punctate bodies were both confirmed by infection with E1A-negative recombinant adenovirus vectors expressing either IE1 or IE2 alone. These results confirm that transient colocalization with and disruption of PML-associated nuclear bodies by IE1 and continuous targeting to PML-associated nuclear bodies by IE2 are intrinsic properties of these two MIE regulatory proteins, which we suggest may represent critical initial events for efficient lytic cycle infection by HCMV.

Construction and Characterization of Herpes Simplex Virus Type 1 Mutants with Conditional Defects in Immediate Early Gene Expression

The herpes simplex virus type 1 (HSV-1) mutantin1814 contains an insertion mutation in the coding sequence for the virion transactivator protein VP16 and is thus impaired for the activation of immediate early (IE) gene expression. This virus was modified further by introducing the Moloney murine leukemia virus LTR promoter in place of the upstream sequences controlling expression of the IE regulatory protein ICP0, to yield mutantin1820. In almost all cell types tested,in1820 initiated infection less efficiently thanin1814, behaving as if lacking both VP16 and ICP0 functions, but in BHK cellsin1820 was less impaired thanin1814. A rescuant ofin1820 at the VP16 locus,in1825, also exhibited a host range phenotype, initiating replication as efficiently as wild-type HSV-1 in BHK cells but inefficiently in other cell types.In1825 was unable to complement an ICP0 null mutant in restricted cells, demonstrating that the promoter exchange prevented the expression of ICP0 protein in functionally significant amounts. The novel host range properties ofin1820 provided a basis for the construction of additional viruses conditionally impaired for IE gene expression and assessment of their value as prototype vectors. Production of an HSV-1 mutant multiply defective in the expression of IE gene products was achieved by introduction of the temperature-sensitive mutation of HSV-1tsK, which inactivates the IE transcription activator ICP4 at nonpermissive temperatures, intoin1820 to producein1820K. This mutant could be propagated effectively in BHK cells at 31° but was effectively devoid of the major regulators ICP0, ICP4, and VP16 in other cells types at 38.5°. Cultures could withstand infection with 5 PFU ofin1820K per cell without detectable cytopathology and could be reseeded to form colonies at approximately 90% efficiency. A derivative ofin1820K containing theEscherichia colilacZ gene controlled by the human cytomegalovirus (HCMV) major IE promoter expressed low but detectable levels of β-galactosidase in almost all cells after infection of cultures at 5 PFU per cell and incubation at 38.5°. Cultures infected with 5 PFU per cell of anin1820K derivative expressing neomycin phosphotransferase (npt) controlled by the HCMV IE promoter were resistant to killing by the antibiotic G418 for up to 3 days, and cell survival correlated with the retention of functional levels of npt. Mutants based onin1820K can thus express foreign gene products in virtually all cells in a culture under conditions in which cytotoxicity is eliminated, demonstrating that progressive reduction of IE gene expression is an important step in the design of HSV-1-derived vectors.

Cell type-specific expression in brain cell cultures from a short human cytomegalovirus major immediate early promoter depends on whether it is inserted into herpesvirus or adenovirus vectors.

Expression from a short human cytomegalovirus (HCMV) major immediate early (IE) promoter-enhancer was tested in three different virus vectors: recombinant adenovirus (Ad), recombinant herpes simplex virus type 1 (HSV-1) and HSV-1-derived amplicon vectors. The HCMV major IE promoter-enhancer within a replication-deficient recombinant Ad vector was shown to produce cell-specific expression in rat nervous system cell cultures. Recombinant Ad entered all cell types examined but the HCMV major IE promoter was silent in primary cultures of neocortical neurons and Schwann cells, although it drove transgene expression in astrocytes and fibroblasts. Moreover, in neurons and Schwann cells, expression from the HCMV major IE promoter-enhancer in the replication-deficient Ad vector was activated by superinfection with HSV-1, replication-competent Ad and HCMV. The HCMV major IE promoter-enhancer was active in neurons when inserted into HSV-1 recombinant vectors. Further experiments with HSV-1-derived amplicons strongly suggested that an IE protein was responsible for the activation of HCMV major IE-induced expression in neurons. This demonstrates that the activity of the HCMV major IE promoter-enhancer element can depend on the expression of other genes encoded in the virus vector backbone within which it is inserted, and that it can function in a neuronal cell type-specific manner when inserted into a replication-deficient Ad vector.

Effect of a modulator deletion on transcription of the human cytomegalovirus major immediate-early genes in infected undifferentiated and differentiated cells.

Differentiation-dependent expression of the human cytomegalovirus (HCMV) major immediate-early (MIE) genes, encoding IE1 and IE2, may partly govern virus replication in monocytic THP-1 and embryonal carcinoma (Tera-2) cells. The modulator of the MIE promoter was shown previously in transient transfection assays to repress transcription from promoter segments in undifferentiated THP-1 and Tera-2 cells but not in differentiated cells. To determine the biological importance of these findings, we constructed a recombinant HCMV (r delta MSVgpt) without a modulator. In comparison to wild-type (WT) virus, r delta MSVgpt exhibits a slight delay in growth in human fibroblasts, but there is no appreciable change in IE1 and IE2 transcription. Moreover, there is no appreciable change in the early/late kinetics of transcription of RNAs colinear with the predicted UL128 coding region, which is adjacent to the modulator, although the size distribution and abundance of these RNAs are altered. In infected undifferentiated THP-1 and Tera-2 cells, WT and r alpha MSVgpt viruses produce minimal but comparable amounts of IE1 RNAs. The genomes of both viruses are detectable in similar amounts within these undifferentiated cells. Induction of cellular differentiation before infection overcomes the block in MIE gene transcription. WT and r alpha MSVgpt infections of differentiated THP-1 cells produce similar levels of IE1 and IE2 RNAs. Thus, differentiation-dependent control of MIE gene transcription involves regulatory mechanisms other than the modulator. Possible alternative functions of the modulator are discussed.